Yeast Pyruvate Kinase: A Mutant Form Catalytically Insensitive to Fructose 1,6-Bisphosphate

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Abstract
The characteristic properties of an altered form of pyruvate kinase [EC 2.7.1.40] from a mutant of Saccharomyces cerevisiae are described. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity, but is stabilized in its presence, both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have s20,w values of 8.9 and 8.6 S, respectively. The mutant with the pyruvate kinase insensitive to fructose 1,6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole C source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.